3-deoxy-d-manno-2-octulosonic acid (KDO) is a component of rhamnogalacturonan II,a pectic polysaccharide in the primary cell walls of plants

3-Deoxy-d-manno-2-octulosonic acid (KDO), a sugar previously presumed to occur only as a glycosyl residue in polysaccharides produced by Gram-negative bacteria, was found to be a component of the cell walls of higher plants. In the form of the disaccharide α-l-Rhap-(1→5)-d-KDO, KDO was released by mild hydrolysis with acid from the purified cell wall polysaccharide rhamnogalacturonan II. KDO was shown to be present in purified cell walls of several plants, including dicots, a monocot, and a gymnosperm. Improved methods for detecting and quantitating KDO residues in polysaccharides were developed during this investigation.     

G.L.C.-M.S. of partially methylated and acetylated derivatives of 3-deoxyoctitols

Partially methylated and acetylated 3-deoxyoctitols were prepared from derivatives of 3-deoxy-d-manno-2-octulosonic acid (KDO), and identified as the d-glycero-d-talo and d-glycero-d-galacto isomers by g.l.c.-m.s. Mono- and oligosaccharide derivatives of KDO were subjected in sequence to methylation, carboxyl-reduction, hydrolysis, carbonyl-reduction, and acetylation to yield 1,2,6-tri-O-acetyl-3-deoxyoctitol derivatives. Carboxyl-reduction and then methylation gave the series of 2,6-di-O-acetyl derivatives. Oligosaccharides with KDO at the reducing end, e.g., β-d-ribofuranosyl-(1→7)-KDO, α-l-glycero-d-manno-heptopyranosyl-(1→5)-KDO, and α-KDOp-(2→4)-KDO, yielded, after carbonyl-reduction, methylation, carboxyl-reduction, hydrolysis, and acetylation, the 1,7-, 1,5-, and 1,4-di-O-acetyl derivatives, whereas remethylation after carboxyl-reduction gave the 7-, 5-, and 4-O-acetyl derivatives of 3-deoxyoctitol. General rules for the fragmentation of 3-deoxyoctitols during e.i.-m.s. were established.     

Two additional bacteriophage-associated glycan hydrolases cleaving ketosidic bonds of 3-deoxy-D-manno-octulosonic acid in capsular polysaccharides of Escherichia coli

Two bacteriophages degrading 3-deoxy-D-manno-2-octulosonic acid-(KDO)-containing capsules of Escherichia coli strains were identified. Using modifications of the thiobarbituric acid assay, it was shown that each phage contains a glycan hydrolase activity cleaving one type of ketosidic linkage of KDO. Thus, the enzyme from phage ψ95 catalyses the hydrolysis of β-octulofuranosidonic linkages of the K95 glycan; and ψ1092, the α-octulopyranosidonic linkages of the K? antigen of E. coli LP1092. No cross-reactivity of the phage enzymes with other KDO-containing capsular polysaccharides was observed.     

Localization of the enzyme 3-deoxy-d-manno-2-octulosonic acid aldolase

Summary Several strains of Gram-negative microorganisms were screened for maximum 3-deoxy-d-manno-2-octulosonic acid (KDO) aldolase (EC 4.1.2.23) activity. Although this enzyme has been noted to be inducible on special medium, no induction was found. By centrifugation studies the KDO aldolase was found to be localized in the cell wall or membrane fraction. The enzyme activity was very susceptible to small amounts of detergent in solution. Offprint requests to: M.-R. Kula     

Quantitation of l-glycero-d-manno-heptose and 3-deoxy-d-manno-octulosonic acid in rough core lipopolysaccharides by partition chromatography

A partition chromatographic procedure utilizing a cationic exchange resin column in the Li⁺ form and 90% ethanol as the mobile phase was employed to quantify 3-deoxy-d-manno-octulosonic acid (KDO) and l-glycero-d-manno-heptose in the lipopolysaccharides (LPS) of R_e and R_dP^? rough mutants of Salmonella minnesota. In a standard mixture of monosaccharides, KDO eluted shortly after the void volume and heptose eluted after the neutral hexoses. Mild acid treatment of either the R_e or R_dP^? LPS with 0.16 n methanesulfonic acid in the presence of Dowex 50-X8 resin (H⁺ form) released more than 80% of the KDO residues within 15 min. The heptose of the R_dP^? LPS, first detected after 90 min of hydrolysis, increased gradually to a maximum level at 12 h. A secondary gradual increase in KDO became apparent during the heptose release. The weight contents of these two monosaccharides based upon aheir maximum values detected during hydrolysis were 20.3 ± 0.6% KDO, for the R_e LPS, and 13.8 ± 0.4% KDO and 12.0 ± 0.4% heptose, for the R_dP^? LPS. The relationship between the kinetics of release of KDO and heptose and the nature of the linkages involving these two monosaccharides are discussed.     

Specificity and mutational analysis of the metal-dependent 3-deoxy-d-manno-octulosonate 8-phosphate synthase from Acidithiobacillus ferrooxidans

3-Deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between phosphoenol pyruvate and d-arabinose 5-phosphate to generate KDO8P. This reaction is part of the biosynthetic pathway to 3-deoxy-d-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Two distinct groups of KDO8PSs exist, differing by the absolute requirement of a divalent metal ion. In this study Acidithiobacillus ferrooxidans KDO8PS has been expressed and purified and shown to require a divalent metal ion, with Mn²⁺, Co²⁺ and Cd²⁺ (in decreasing order) being able to restore activity to metal-free enzyme. Cd²⁺ significantly enhanced the stability of the enzyme, raising the T_m by 14 °C. d-Glucose 6-phosphate and d-erythrose 4-phosphate were not substrates for A. ferrooxidans KDO8PS, whereas 2-deoxy-d-ribose 5-phosphate was a poor substrate and there was negligible activity with d-ribose 5-phosphate. The ²⁴³AspGlyPro²⁴⁵ motif is absolutely conserved in the metal-independent group of synthases, but the Gly and Pro sites are variable in the metal-dependent enzymes. Substitution of the putative metal-binding Asp243 to Ala in A. ferrooxidans KDO8PS gave inactive enzyme, whereas substitutions Asp243Glu or Pro245Ala produced active enzymes with altered metal-dependency profiles. Prior studies indicated that exchange of a metal-binding Cys for Asn converts metal-dependent KDO8P synthase into a metal-independent form. Unexpectedly, this mutation in A. ferrooxidans KDO8P synthase (Cys21Asn) gave inactive enzyme. This finding, together with modest activity towards 2-deoxy-d-ribose 5-phosphate suggests similarities between the A. ferrooxidans KDO8PS and the related metal-dependent 3-deoxy-d-arabino-heptulosonate phosphate synthase, and highlights the importance of the AspGlyPro loop in positioning the substrate for effective catalysis in all KDO8P synthases.     

Structure of the 3-deoxy-d-manno-octulosonic acid-containing polysaccharide (K6 antigen) from escherichia coli LP 1092

The acidic polysaccharide (K6) antigen from Escherichia coli LP 1092 contains d-ribose and 3-deoxy-d-manno-octulosonic acid in the molar ratio of 2:1, respectively. Spectroscopic data (¹³C- and ¹H-n.m.r.), methylation analyses, and periodate oxidation indicate that the polysaccharide is composed of the foregoing components essentially in the following trisaccharide sequence: →2)-β-d-Ribf-(1→2)-β-d-Ribf-(1→7)-α-d-KDO-(2→The polysaccharide also contains O-acetyl substituents (～0.2–0.3 mol per KDO residue).     

Characterization of 3-deoxy-D-manno-octulosonic acid as a component of the capsular polysaccharide antigen from Neisseria meningitidis serogroup 29-e

3-deoxy-$\text{D}$-$\text{manno}$ acid (KDO) has been characterised as the major component (53%) of the capsular polysaccharide antigen of $\text{N}\text{. meningitidis}$ serogroup 29-e. This is the first reported occurrence of KDO in any biological polymer other than its well established occurrence in the lipopolysaccharides of gram-negative bacteria.     

2-KETO-SUGAR ACIDS IN GREEN FLAGELLATES: A CHEMICAL MARKER FOR PRASINOPHYCEAN SCALES1,2

The chemical composition of cell walls (thecae) of three taxa of scaly green flagellates (Prasinophyceae) was investigated. The theca of Tetraselmis striata, Tetraselmis tetrathele, and Scherffelia dubia consists mainly of carbohydrate (80% of dry weight), with proteins (5%), calcium (4%), and sulfate (6%) as minor components. The principal sugars (60% of dry weight) are the 2-keto-sugar acids 3-deoxy-manno-2-octulosonic acid (KDO), 3-deoxy-manno-5-O-methyl-2-octulosonic acid (5OMeKDO), and 3-deoxy-lyxo-2-heptulosaric acid (DHA). Arabinose, gulose, galactose, galacturonic acid, and in S. dubia, xylose and rhamnose were also found. Examination of scale preparations from Mantoniella squamata, Mesostigma viride, Pyramimonas amylifera, and Nephroselmis olivacea revealed that the 2-keto-sugar acids were always associated with the presence of typical prasinophycean scales on the cell surface. In contrast, 2-keto-sugar acids were not detected in the cell wall of Chlamydomonas reinhardtii nor in polymer preparations from the culture medium of Chlamydomonas reinhardtii, Dunaliella bioculata, Dunaliella primolecta, Asteromonas gracilis, Hafniomonas reticulate, Pedinomonas tuberculata, Monomastix sp., and Micromonas pusilla. We conclude that 2-keto-sugar acids are chemical markers for prasinophycean scales.     

Characterization of an extracellular polysaccharide from a xanthomonas species

An extracellular polysaccharide containing glucose, mannose, D-manno-octulosonic acid (KDO), an unidentified component (X), and acetyl groups in the molar ratio of 1.3:3.8:1.6:1.1:2.9, was obtained from the incubated medium of a Xanthomonas species. The extracellular polysaccharide contained traces of phosphate and nitrogen but no lipid. Mild hydrolysis with 0.025M sulfuric acid released all of the KDO in the polysaccharide and a KDO-free product was obtained, which on hydrolysis with 0.05M sulfuric acid, gave mainly an oligosaccharide containing glucose, mannose, and X in molar ratio of 1:1:1. The reducing end-group of this oligosaccharide was X, and other hexose residues were linked (1 → 4). Compound X seems to be a 6-deoxyhexose that differs from fucose and rhamnose.     

Homo-C-nucleoside analogs III. Studies on the base-catalyzed dehydrative cyclization of 4-(d-manno-pentitol-1-yl)-2-phenyl-2H-1,2,3-triazole

Treatment of 4-(d-manno-pentitol-1-yl)-2-phenyl-2H-1,2,3-triazole with one molar equivalent of 2,4,6-triisopropylbenzenesulfonyl chloride (TIBSCl) in pyridine solution afforded the homo-C-nucleoside analog; 4-(2,5-anhydro-d-manno-pentitol-1-yl)-2-phenyl-2H-1,2,3-triazole in 54% yield and 4-(α-d-arabinopyranosyl)-2-phenyl-2H1,2,3-triazole analog in 3% yield. The 4-(5-O-triisopropylbenzenesulfonyl)-d-manno-pentitol-1-yl)-2-phenyl-2H-1,2,3-triazole analog was isolated as an intermediate and identified as its tetra-O-acetyl derivative. The 4-(5-chloro-5-deoxy-d-manno-pentitol-1-yl)-2-phenyl-2H-1,2,3-triazole analog was isolated as a byproduct. The structure and anomeric configuration of the products were determined by acylation, NMR spectroscopy, and mass spectrometry.     

Synthese von O-(3-desoxy-α-d-manno-2-octulopyranosylonsäure)-(2→4)-O-(3-desoxy-α-d-manno-2-octulopyranosylonsäure)- (2→6)-O-(2-amino-2-desoxy-β-d-glucopyranosyl)-(1→6)-2-amino-2-desoxy-d-glucopyranose

Benzyl-3-O-benzyl-2-benzyloxycarbonylamino-6-O-[2-benzyloxycarbonyl-amino-2-deoxy-3,4-O-(tetraisopropyldisiloxane-1,3-diyl)- β-d-glucopyranosyl]-2-deoxy-α-d-glucopyranoside was coupled with methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-α-d-manno-2-octulopyranosyl bromide)onate (13) to yield the α-glycosidically linked trisaccharide. After deacetylation and selective introduction of a second 7′,8′-O-tetraisopropyldisiloxane group, a further glycosidation reaction with 13 led regioselectively to the tetrasaccharide benzyl O-[methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-α-d-manno-2-octulopyranosyl)onate]-(2→4)-O-{methyl [3-deoxy-7,8-O-(tetraisopropyldisiloxane-1,3-diyl)-α-d-manno-2-octulopyranosyl]-onate}-(2→6)-O- [2-benzyloxycarbonylamino-2-deoxy-3,4-O-(tetraisopropyldisiloxane-1,3-diyl)-β-d-glucopyranosyl]- (1→6)-3-O-benzyl-2-benzyloxycarbonyl-amino-2-deoxy-α-d-glucopyranoside. A series of deblocking steps gave O-(3-deoxy-α-d-manno-2-octulopyranosylonic acid)-(2→4)-O-(3-deoxy-α-d-manno-2-octulopyranosylonic acid)- (2→6)-O-(2-amino-2-deoxy-β-d-glucopyranosyl)-(1→6)-2-amino-2-deoxy-d-glucopyranose which was identical with a tetrasaccharide that had been isolated by hydrazinolysis of the lipopolysaccharide from Salmonella minnesota R 595. Hence, synthetic proof is provided for the linkages in this part of the inner core region of lipopolysaccharides.     

Colorimetric estimation of 3-deoxy-D-manno-octulosonic acid in oligosaccharides with diphenylamine

A method for the colorimetric estimation of 3-deoxy-d-manno-octulosonic acid with diphenylamine is described. The aldulosonic acid can be estimated in the presence of a tenfold excess of other sugars.     

Syntheses of repeating units of Escherichia coli capsular polysaccharides containing d-ribose and 3-deoxy-d-manno-2-octulosonic acid (KDO)

The oligosaccharides, sodium (methyl 3-deoxy-7-O-β-d-ribofuranosyl-β-d-manno-2-octulopyranosid)onate, methyl 2-O-β-d-ribofuranosyl-β-d-ribofuranoside, and the anomeric sodium [methyl 3-deoxy-7-O-(2-O-β-d-ribofuranosyl-β-d-ribofuranosyl)-α- and -β-d-manno-2-octulopyranosid]onate were prepared from 1-O-acetyl-2,3,5-tri-O-benzoyl-β-d-ribofuranose and the anomeric methyl (methyl 8-O-benzyl-4,5-O-carbonyl-3-deoxy-α- and -β-d-manno-2-octulopyranosid)onate in high purity and in acceptable over-all yields. They constitute a first series of model compounds for spectroscopic and immunochemical studies of the capsular polysaccharides from Escherichia coli strains LP 1092 and K 23. The essential, interglycosidic linkages [β-d-Ribf-(1→7)-α- or -β-d-dOclA, and β-d-Ribf-(1→2)-β-d-Ribf] were formed by a modification of the silver triflate procedure using appropriate d-ribofuranosyl bromide derivatives. The constitutional and configurational assignments were based on the 250-MHz ¹H-n.m.r.-spectra of protected derivatives of the oligosaccharides.     

Analytical studies of lipopolysaccharide and its derivatives from Salmonella minnesota R595. III. Reappraisal of established methods

Established methods for analysis of components of lipopolysaccharides were assessed. Optimal release of glucosamine from lipopolysaccharide occurs after hydrolysis in 6 M hydrochloric acid at 100°C for 4 h and fatty acids are best released by treatment with boron trifluoride/methanol at 100°C for 6 h. The semicarbazide assay for 3-deoxy-d-manno-octulosonic acid was modified to give results comparable to those from the periodate/thiobarbituric acid method. It was concluded that each molecule of lipopolysaccharide from Salmonella minnesota R595 contains two octulosonic acid residues and only four fatty acids, on average. There are two amide-linked hydroxyacids, together with, on average, 0.5 residues of ester hydroxyacid and a total of 1.5 residues of ester-linked normal fatty acids. This conclusion differs from the accepted view of Salmonella lipid A, but is supported by NMR results.     

Reversing Evolution: Re-establishing Obligate Metal Ion Dependence in a Metal-independent KDO8P Synthase

Two distinct groups of 3-deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS), a key enzyme of cell-wall biosynthesis, differ by their requirement for a divalent metal ion for enzymatic activity. The unique difference between these groups is the replacement of the metal-binding Cys by Asn. Substitution of just this Asn for a Cys in metal-independent KDO8PS does not create the obligate metal-ion dependency of natural metal-dependent enzymes. We describe how three or four mutations of the metal-independent KDO8PS from Neisseria meningitidis produce a fully functional, obligately metal-dependent KDO8PS. For the substitutions Asn23Cys, Asp247Glu (this Asp binds to the metal ion in all metal-dependent KDO8PS) and Pro249Ala, and for double and triple combinations, mutant enzymes that contained Cys in place of Asn showed an increase in activity in the presence of divalent metal ions. However, combining these mutations with substitution by Ser of the Cys residue in the conserved ²⁴⁶CysAspGlyPro²⁴⁹ motif of metal-independent KDO8PS created enzymes with obligate metal dependency. The quadruple mutant (Asn23Cys/Cys246Ser/Asp247Glu/Pro249Ala) showed comparable activity to wild-type enzymes only in the presence of metal ions, with maximum activity with Cd²⁺, the metal ion that is strongly inhibitory at micromolar concentrations for the wild-type enzyme. In the absence of metal ions, activity was barely detectable for this quadruple mutant or for triple mutants bearing both Cys246Ser and Asn23Cys mutations. The structures of NmeKDO8PS and its Asn23Cys/Asp247Glu/Pro249Ala and quadruple mutants at pH 4.6 were characterized at resolutions better than 1.85 Å. Aged crystals of the Asn23Cys/Asp247Glu/Pro249Ala mutant featured a Cys23-Cys246 disulfide linkage, explaining the spectral bleaching observed when this mutant was incubated with Cu²⁺. Such bleaching was not observed for the quadruple mutant. Reverse evolution to a fully functional obligately metal-dependent KDO8PS has been achieved with just three directed mutations for enzymes that have, at best, 47% identity between metal-dependent and metal-independent pairs.     

The synthesis of 5-amino-2,6-anhydro-5-deoxy-D-glycero-D-gulo-Heptonic acid and its polycondensation to oligomers

5-Amino-2,6-anhydro-5-deoxy-D-glycero-D-gulo-heptonic acid has been synthesized by conventional introduction of an amino function via azide displacement, starting with a suitable derivative of 2,6-anhydro-D-glycero-L-manno-heptonic acid. The amino acid was converted into the methyl ester hydrochloride which, in methanolic sodium methoxide, gave oligomeric and polymeric amides, depending on the conditions applied. Four oligomeric esters, as well as the corresponding N-(2,4-dinitrophenyl) derivatives of the amino acids, could be separated by paper chromatography. The oligomers could be saponified under mild, basic conditions.     

Synthesis of a neoglycoconjugate containing a Chlamydophila psittaci-specific branched Kdo trisaccharide epitope

The branched Kdo trisaccharide sodium (3-deoxy-α-d-manno-oct-2-ulopyranosyl)onate-(2→8)-[sodium (3-deoxy-α-d-manno-oct-2-ulopyranosyl)onate-(2→4)]-sodium (allyl 3-deoxy-α-d-manno-oct-2-ulopyranosid)onate has been prepared utilizing the regioselective glycosylation of the C-7, C-8 diol entity of a Kdo monosaccharide acceptor with a Kdo bromide donor followed by the attachment of the third Kdo unit to O-4 of the disaccharide intermediate. Deacetylation and hydrolysis of the methyl ester groups furnished the trisaccharide allyl glycoside which was converted into the corresponding 3-(2-aminoethylthio)propyl glycoside. Subsequent covalent attachment to bovine serum albumin furnished a neoglycoconjugate serving as an antigen for the induction of Chlamydophila psittaci-specific monoclonal antibodies.     

Determination of 3-deoxy-D-manno-octulosonic acid (KDO), N-acetylneuraminic acid, and their derivatives by ion-exchange liquid chromatography

A liquid chromatography (1.6 MPa) system for the analysis of 3-deoxy-D-manno-2-octulosonic acid (KDO), N-acetylneuraminic acid (Neu5Ac), methyl alpha- and beta-glycosides of Neu5Ac and KDO, alpha-heptosyl-(1----5)-KDO, various sialyllactoses, alpha-KDO-(2----4)-KDO, alpha-KDO-(2----4)-KDO methyl alpha-glycoside, beta-KDO-(2----4)-KDO methyl beta-glycoside, D-glucuronic acid, D-glucurono-3,6-lactone, and D-galacturonic acid has been developed. Separation was achieved within 10 and 30 min by the use of a small column filled with a strongly basic, anion-exchange resin, Aminex A-29, and 0.75 or 10mM sodium sulfate solutions as mobile phases. This method allowed the determination of KDO and sialic acids in amounts of 100 ng (0.5 nmol) and 200 pg (0.6 pmol), respectively.     

Lipopolysaccharides of Thiobacillus species containing lipid A with 2,3-diamino-2,3-dideoxyglucose

Lipopolysaccharides were isolated from two strains of Thiobacillus ferrooxidans and one strain each of Thiobacillus thiooxidans, Thiobacillus novellus and Thiobacillus sp. IFO 14570. Neutral sugars, 2-keto-3-deoxyoctonate, fatty acids and the rare 2,3-diamino-2,3-dideoxyglucose were detected in all lipopolysaccharides. Lipopolysaccharides of both T. ferrooxidans strains contained l-glycero-d-manno-heptose, whereas that of T. thiooxidans contained both l-glycero-d-manno-heptose and d-glycero-d-manno-heptose. On the other hand, heptoses were absent in lipopolysaccharides of T. novellus and Thiobacillus sp. IFO 14570. Lipid A of T. ferrooxidans and T. thiooxidans contained both glucosamine and 2,3-diamino-2,3-dideoxyglucose, in contrast, lipid A of T. novellus and the Thiobacillus sp. IFO 14570 most likely contain only 2,3-diamino-2,3-dideoxyglucose as backbone sugar. Deoxycholate polyacrylamide gel electrophoresis revealed S-type character for all lipopolysaccharides studied. The significance of the lipopolysaccharide composition for taxonomic and phylogenetic questions with regard to thiobacilli is discussed.Abbreviations DAG 2,3-diamino-2,3-dideoxyglucose - DOC sodium deoxycholate - GC gas-liquid chromatography - GC/MS gas-liquid chromatography/mass spectrometry - d,d-Heptose d-glycero-d-manno-heptose - l,d-Heptose l-glycero-d-manno-heptose - KDO 2-keto-3-deoxyoctonate - LPS lipopolysaccharide - 3-OH-14:0 3-hydroxy-tetradecanoic acid - PAGE polyacrylamide gel electrophoresis - PCP phenol-chloroform-petroleum ether